The invention relates to derivatives of poly(ethylene glycol) and related hydrophilic polymers, to methods for their synthesis, and to surfaces and molecules modified by these polymers.
Covalent attachment of the hydrophilic polymer poly(ethylene glycol), abbreviated PEG, also known as poly(ethylene oxide), abbreviated PEO, to molecules and surfaces is of considerable utility in biotechnology and medicine. In its most common form, PEG is a linear polymer terminated at each end with hydroxyl groups:
HOxe2x80x94CH2CH2Oxe2x80x94(CH2CH2O)nxe2x80x94CH2CH2xe2x80x94OH
The above polymer, alpha-, omega-dihydroxylpoly(ethylene glycol), can be represented in brief form as HOxe2x80x94PEGxe2x80x94OH where it is understood that the xe2x80x94PEGxe2x80x94 symbol represents the following structural unit:
xe2x80x94CH2CH2Oxe2x80x94(CH2CH2O)nxe2x80x94CH2CH2xe2x80x94
where n typically ranges from about 3 to about 4000.
PEG is commonly used as methoxy-PEGxe2x80x94OH, or mPEG in brief, in which one terminus is the relatively inert methoxy group, while the other terminus is a hydroxyl group that is subject to ready chemical modification. The structure of mPEG is given below.
xe2x80x83CH3Oxe2x80x94(CH2CH2O)nxe2x80x94CH2CH2xe2x80x94OH
The copolymers of ethylene oxide and propylene oxide are closely related to PEG in their chemistry, and they can be substituted for PEG in many of its applications.
HOxe2x80x94CH2CHRO(CH2CHRO)nCH2CH2xe2x80x94OH
where Rxe2x95x90H or alkyl, such as CH3.
PEG is a polymer having the properties of solubility in water and in many organic solvents, lack of toxicity, and lack of immunogenicity. One use of PEG is to covalently attach the polymer to insoluble molecules to make the resulting PEG-molecule xe2x80x9cconjugatexe2x80x9d soluble. For example, it has been shown that the water-insoluble drug paclitaxel, when coupled to PEG, becomes water-soluble. Greenwald, et al., J. Org. Chem., 60:331-336 (1995).
To couple PEG to a molecule, such as a protein, it is often necessary to xe2x80x9cactivatexe2x80x9d the PEG to prepare a derivative of the PEG having a functional group at the terminus. The functional group can react with certain moieties on the protein, such as an amino group, thus forming a PEG-protein conjugate. Many activated derivatives of PEG have been described. An example of such an activated derivative is the succinimidyl succinate xe2x80x9cactive esterxe2x80x9d:
CH3Oxe2x80x94PEGxe2x80x94O2Cxe2x80x94CH2CH2xe2x80x94CO2xe2x80x94NS
where NSxe2x95x90
Hereinafter, the succinimidyl active ester moiety will be represented as xe2x80x94CO2xe2x80x94NS.
As applications of PEG chemistry have become more sophisticated, there has been an increasing need for heterofunctional PEGs, that is, PEGs bearing dissimilar terminal groups:
xe2x80x83Xxe2x80x94PEGxe2x80x94Y
where X and Y are different groups. Such heterobifunctional PEGs bearing appropriate functional groups may be used to link the PEG to surfaces or biologically active molecules, with the other terminus attached, for example, to a biologically active molecule, a liposome, or a biosensor.
It is desirable in the biotechnical arts to continually develop activated polymers suitable for conjugation with one or more of various substances, including other polymers, peptides, proteins, carbohydrates, oligonucleotides, lipids, liposomes, cells, drugs, surfaces, and other biologically active moieties. Additionally, it would be advantageous to develop activated polymers that can be used for targeting or extended release formulations.
The invention utilizes hydroxyapatite surfaces, such as bone, for the delivery of biologically active agents with sustained lifetime within the body. Polyethylene glycol is often covalently attached to biologically active molecules to extend its circulation half-life, but the residence time of some conjugates remains suboptimal. There are many biologically active agents, both polypeptides and small drug molecules, that would benefit from the extended residence time within the body and targeting of hydroxyapatite surfaces, such as bone, provided by the invention described more filly below.
The invention provides an isolatable, activated hydroxyapatite-targeting polymeric structure comprising a linear or branched water-soluble and non-peptidic polymer backbone having at least two termini, a first terminus being covalently bonded to a hydroxyapatite-targeting moiety and a second terminus covalently bonded to a chemically reactive group or a protected chemically reactive group. For example, the hydroxyapatite-targeting moiety can be selected from the group consisting of tetracycline, calcein, bisphosphonates, polyaspartic acid, polyglutamic acid, and aminophosphosugars. The chemically reactive group or protected chemically reactive group is preferably selected from the group consisting of hydroxyl, protected hydroxyl, active ester, active carbonate, acetal, aldehyde, aldehyde hydrates, alkenyl, acrylate, methacrylate, acrylamide, active sulfone, amine, protected amine, hydrazide, protected hydrazide, thiol, protected thiol, carboxylic acid, protected carboxylic acid, isocyanate, isothiocyanate, maleimide, vinylsulfone, dithiopyridine, vinylpyridine, iodoacetamide, epoxide, glyoxals, diones, mesylates, tosylates, and tresylate. The polymer backbone may comprise, for example, poly(alkylene glycol), poly(oxyethylated polyol), poly(olefinic alcohol), poly(vinylpyrolidone), poly(hydroxypropylmethacrylamide), poly(xcex1-hydroxy acid), poly(vinyl alcohol), polyphosphazene, polyoxazoline, and copolymers, terpolymers, derivatives and mixtures thereof.
By reacting the chemically reactive group with a biologically active agent, the hydroxyapatite-targeting polymers of the invention can be used to tether a biologically active agent to a surface, such as a bone surface. Methods for preparation of the hydroxyapatite-targeting polymers, and biologically active conjugates thereof, are also provided.
In one embodiment, the invention provides a method of utilizing a bone surface in a bone-containing organism, such as a mammal, as a reservoir for a releasable biologically active agent. The method includes providing a hydroxyapatite-targeting, biologically active polymeric structure comprising a linear or branched water-soluble and non-peptidic polymer backbone having at least two termini, a first terminus being covalently bonded to a hydroxyapatite-targeting moiety and a second terminus covalently bonded to a biologically active agent through a linker, wherein at least one of the polymer backbone and the linker comprise a hydrolytically or enzymatically degradable linkage. A therapeutically effective amount of the polymeric structure is administered to a bone-containing organism such that at least a portion of the polymeric structure is bonded to a bone surface by the hydroxyapatite-targeting moiety. Preferably, the hydrolytically or enzymatically degradable linkage is selected from the group consisting of carbonate, carboxylate ester, phosphoester, orthoester, acetal, carbamate, disulfide, and peptide. The hydroxyapatite-targeting polymeric structure with the releasable biologically active agent will initially target bone or bone marrow surfaces within the organism, thereby using the bone surface as a reservoir or depot. The biologically active agent will be released into the organism over time as the degradable linkage degrades.
Thus, the invention allows a biologically active agent to be anchored to a hydroxyapatite surface in vivo and delivered over time to other parts of the organism for treatment of disease. In this manner, the residence time of the biologically active agent could be extended and the efficacy of the treatment improved. In addition, the activated polymer derivatives of the invention are isolatable such that the polymers can be separated and purified prior to attachment to a biologically active agent, thereby increasing yield and purity of the biologically active polymers.
The terms xe2x80x9cfunctional groupxe2x80x9d, xe2x80x9cactive moietyxe2x80x9d, xe2x80x9cactivating groupxe2x80x9d, xe2x80x9creactive sitexe2x80x9d, xe2x80x9cchemically reactive groupxe2x80x9d and xe2x80x9cchemically reactive moietyxe2x80x9d are used in the art and herein to refer to distinct, definable portions or units of a molecule. The terms are somewhat synonymous in the chemical arts and are used herein to indicate that the portions of molecules that perform some function or activity and are reactive with other molecules.
The term xe2x80x9clinkagexe2x80x9d or xe2x80x9clinkerxe2x80x9d is used herein to refer to groups or bonds that normally are formed as the result of a chemical reaction and typically are covalent linkages. Hydrolytically stable linkages means that the linkages are substantially stable in water and do not react with water at useful pHs, e.g., under physiological conditions for an extended period of time, preferably indefinitely. Hydrolytically unstable or degradable linkages means that the linkages are degradable in water or in aqueous solutions, including for example, blood. Enzymatically unstable or degradable linkages means that the linkage can be degraded by one or more enzymes.
As used herein, the term xe2x80x9ccompoundxe2x80x9d is intended to refer to a chemical entity, whether in the solid, liquid or gaseous phase, and whether in a crude mixture or purified and isolated. The terms xe2x80x9calkyl,xe2x80x9d xe2x80x9calkene,xe2x80x9d and xe2x80x9calkoxyxe2x80x9d include straight chain and branched alkyl, alkene, and alkoxy, respectively. The term xe2x80x9clower alkylxe2x80x9d refers to C1-C6 alkyl. The term xe2x80x9calkoxyxe2x80x9d refers to oxygen substituted alkyl, for example, of the formulas xe2x80x94OR or xe2x80x94ROR1, wherein R and R1 are each independently selected alkyl. The terms xe2x80x9csubstituted alkylxe2x80x9d and xe2x80x9csubstituted alkenexe2x80x9d refer to alkyl and alkene, respectively, substituted with one or more non-interfering substituents, such as but not limited to, C3-C6 cycloalkyl, e.g., cyclopropyl, cyclobutyl, and the like; acetylene; cyano; alkoxy, e.g., methoxy, ethoxy, and the like; lower alkanoyloxy, e.g., acetoxy; hydroxy; carboxyl; amino; lower alkylamino, e.g., methylamino; ketone; halo, e.g. chloro or bromo; phenyl; substituted phenyl, and the like. The term xe2x80x9chalogenxe2x80x9d includes fluorine, chlorine, iodine and bromine.
xe2x80x9cArylxe2x80x9d means one or more aromatic rings, each of 5 or 6 carbon atoms. Multiple aryl rings may be fused, as in naphthyl or unfused, as in biphenyl. Aryl rings may also be fused or unfused with one or more cyclic hydrocarbon, heteroaryl, or heterocyclic rings. xe2x80x9cSubstituted arylxe2x80x9d is aryl having one or more non-interfering groups as substituents.
xe2x80x9cNon-interfering substituentsxe2x80x9d are those groups that yield stable compounds. Suitable non-interfering substituents or radicals include, but are not limited to, halo, C1-C10 alkyl, C2-C10 alkenyl, C2-C10 alkynyl, C1-C10 alkoxy, C7-C12 aralkyl, C7-C12 alkaryl, C3-C10 cycloalkyl, C3-C10 cycloalkenyl, phenyl, substituted phenyl, toluoyl, xylenyl, biphenyl, C2-C12 alkoxyalkyl, C7-C12 alkoxyaryl, C7-C12 aryloxyalkyl, C6-C12 oxyaryl, C1-C6 alkylsulfinyl, C1-C10 alkylsulfonyl, xe2x80x94(CH2)mxe2x80x94Oxe2x80x94(C1-C10 alkyl) wherein m is from 1 to 8, aryl, substituted aryl, substituted alkoxy, fluoroalkyl, heterocyclic radical, substituted heterocyclic radical, nitroalkyl, xe2x80x94NO2, xe2x80x94CN, xe2x80x94NRC(O)xe2x80x94(C1-C10 alkyl), xe2x80x94C(O)xe2x80x94(C1-C10 alkyl), C2-C10 thioalkyl, xe2x80x94C(O)Oxe2x80x94(C1-C10 alkyl), xe2x80x94OH, xe2x80x94SO2, xe2x95x90S, xe2x80x94COOH, xe2x80x94NR2, carbonyl, xe2x80x94C(O)xe2x80x94(C1-C10 alkyl)xe2x80x94CF3, xe2x80x94C(O)xe2x80x94CF3, xe2x80x94C(O)NR2, xe2x80x94(C1-C10 alkyl)xe2x80x94Sxe2x80x94(C6-C12 aryl), xe2x80x94C(O)xe2x80x94(C6-C12 aryl), xe2x80x94(CH2)mxe2x80x94Oxe2x80x94(CH2)mxe2x80x94Oxe2x80x94(C1-C10 alkyl) wherein each m is from 1 to 8, xe2x80x94C(O)NR2, xe2x80x94C(S)NR2, xe2x80x94SO2NR2, xe2x80x94NRC(O)NR2, xe2x80x94NRC(S)NR2, salts thereof, and the like. Each R as used herein is H, alkyl or substituted alkyl, aryl or substituted aryl, aralkyl, or alkaryl.
The term xe2x80x9camino acidxe2x80x9d includes all essential and non-essential amino acids. Standard amino acid abbreviations known in the art are used herein.
The term xe2x80x9cbiologically active moleculexe2x80x9d, xe2x80x9cbiologically active moietyxe2x80x9d or xe2x80x9cbiologically active agentxe2x80x9d when used herein means any substance which can affect any physical or biochemical properties of a biological organism, including but not limited to viruses, bacteria, fungi, plants, animals, and humans. In particular, as used herein, biologically active molecules include any substance intended for diagnosis, cure mitigation, treatment, or prevention of disease in humans or other animals, or to otherwise enhance physical or mental well-being of humans or animals. Examples of biologically active molecules include, but are not limited to, peptides, proteins, enzymes, small molecule drugs, dyes, lipids, nucleosides, oligonucleotides, cells, viruses, liposomes, microparticles and micelles. Classes of biologically active agents that are suitable for use with the invention include, but are not limited to, antibiotics, fingicides, anti-viral agents, anti-inflammatory agents, anti-tumor agents, cardiovascular agents, anti-anxiety agents, hormones, growth factors, steroidal agents, and the like.
The term xe2x80x9cisolatablexe2x80x9d is intended to mean that the activated polymers can be isolated or separated from other compounds prior to attachment to a biologically active agent. The acceptable level of isolation or purity will depend on various factors, such as the difficulty of purification, the type of contaminating compounds present, etc. By isolating or separating the hydroxyapatite-targeting activated polymers prior to attachment to the biologically active agent, better yield and purity is achieved. In addition, isolation of the polymer derivatives allows qualitative and quantitative analysis of the polymer prior to incorporation of the biologically active agent, which, in turn, increases the overall quality of the biologically active product. Having an isolatable hydroxyapatite-targeting activated polymer available for attachment to biologically active agents can result in a conjugate with a well-defined composition that can easily be conveyed to regulatory authorities. As exemplified in the examples below, the activated polymer derivatives are generally isolated and purified by precipitation followed by solvent extraction and/or chromatography techniques, such as ion-exchange chromatography.
The invention provides an isolatable hydroxyapatite-targeting polymeric structure comprising a linear or branched water-soluble and non-peptidic polymer backbone having at least two termini, a first terminus being covalently bonded to a hydroxyapatite-targeting moiety and a second terminus covalently bonded to a chemically reactive group, wherein the chemically reactive group is protected or unprotected.
The polymer backbone is a substantially non-immunogenic polymer, such as poly(ethylene glycol) (PEG). However, it should be understood that other related polymers are also suitable for use in the practice of this invention and that the use of the term PEG or poly(ethylene glycol) is intended to be inclusive and not exclusive in this respect. Preferably, the polymer backbone has from 2 to about 300 termini.
PEG is typically clear, colorless, odorless, soluble in water, stable to heat, inert to many chemical agents, does not hydrolyze or deteriorate, and is generally nontoxic. Poly(ethylene glycol) is considered to be biocompatible, which is to say that PEG is capable of coexistence with living tissues or organisms without causing harm. More specifically, PEG is non-immunogenic, which is to say that PEG does not tend to produce an immune response in the body. When attached to a molecule having some desirable function in the body, such as a biologically active agent, the PEG tends to mask the agent and can reduce or eliminate any immune response so that an organism can tolerate the presence of the agent. PEG conjugates tend not to produce a substantial immune response or cause clotting or other undesirable effects. PEG having the formula xe2x80x94CH2CH2Oxe2x80x94(CH2CH2O)nxe2x80x94CH2CH2xe2x80x94, where n is from about 3 to about 4000, preferably from about 3 to about 2000, is one useful polymer in the practice of the invention. Preferably, PEG having a molecular weight of from about 200 Da to about 100,000 Da is used as the polymer backbone.
The polymer backbone can be linear or branched. Branched polymer backbones are generally known in the art. Typically, a branched polymer has a central branch core moiety and a plurality of linear polymer chains linked to the central branch core. PEG is commonly used in branched forms that can be prepared by addition of ethylene oxide to various polyols, such as glycerol, pentaerythritol and sorbitol. The central branch moiety can also be derived from several amino acids, such as lysine. The branched polyethylene glycols can be represented in general form as R(xe2x80x94PEGxe2x80x94OH)m in which R represents the core moiety, such as glycerol or pentaerythritol, and m represents the number of arms.
Many other polymers are also suitable for the invention. These polymers can be either in linear form or branched form, and include, but are not limited to, other poly(alkylene glycol), such as poly(propylene glycol) (xe2x80x9cPPGxe2x80x9d), copolymers of ethylene glycol and propylene glycol and the like, poly(oxyethylated polyol), poly(olefinic alcohol), poly(vinylpyrrolidone), poly(hydroxypropylmethacrylamide), poly(xcex1-hydroxy acid), poly(vinyl alcohol), polyphosphazene, polyoxazoline, and copolymers, terpolymers, derivatives and mixtures thereof. Although the molecular weight of each chain of the polymer backbone can vary, it is typically in the range of from about 100 Da to about 100,000 Da, preferably from about 6,000 Da to about 80,000 Da.
Those of ordinary skill in the art will recognize that the foregoing list for ubstantially water soluble non-immunogenic polymer backbone is by no means xhaustive and is merely illustrative, and that all polymeric materials having the qualities described above are contemplated.
The hydroxyapatite-targeting moiety may comprise any moiety capable of binding to, or otherwise exhibiting a chemical affinity for, hydroxyapatite surfaces (i.e. calcium phosphate), such as bone. The hydroxyapatite-targeting moiety is preferably capable of binding to any hydroxyapatite or calcium phosphate surface. In one embodiment, the hydroxyapatite-targeting moiety is selected from the group consisting of tetracycline, calcein, bisphosphonates, polyaspartic acid, polyglutamic acid, and aminophosphosugars. In a particularly preferred embodiment, the hydroxyapatite-targeting moiety is a bisphosphonate.
An example of a bisphosphonate suitable for use with the invention is shown below. 
wherein Y and Z are independently selected from the group consisting of hydrogen, xe2x80x94OH, halogen, aryl, substituted aryl, pyridyl, furanyl, pyrrolidinyl, imidazonyl, C1-C30 alkyl, C1-C30 substituted alkyl, NH2, NHRxe2x80x2, NRxe2x80x22, SH, and SRxe2x80x2, where Rxe2x80x2 is C1-C30 alkyl, C1-C10 alkoxy, aryl or substituted aryl, and W is selected from the group consisting of hydrogen, alkyl, substituted alkyl, aryl, substituted aryl, Na+, and K+. Preferably, Y is xe2x80x94NH(CH2)pxe2x80x94, where p is about 2 to about 6, Z is xe2x80x94OH, and each W is hydrogen. A particularly preferred bisphosphonate is 3-amino-1-hydroxypropane-1,1-diphosphonic acid.
Bisphosphonates are characterized by two carbon-phosphorous bonds, the carbon atom replacing the oxygen in the Pxe2x80x94Oxe2x80x94P (phosphorous-oxygen-phosphorous) bond of pyrophosphate and the Pxe2x80x94Cxe2x80x94P bond conferring resistance to chemical and enzymatic hydrolysis. Different substitutions on the carbon atom have created several different bisphosphonates, each with its own pharmacological properties. Etidronate, which contains a hydroxyl and methyl group substitution on the carbon atom, was the first bisphosphonate with a half-life in bone of greater than 90 days to be used therapeutically. Other more potent bisphosphonates have subsequently been developed, such as alendronate, which has an alkyl amine and hydroxyl group substitution on the carbon atom.
Bisphosphonates have a strong affinity for hydroxyapatite crystals and, in fairly high doses, inhibit calcification of bone in vivo by physicochemical mechanisms. Bisphosphonates are not metabolized, and seem to be absorbed, excreted, and stored unchanged. However, the side chains of some analogs of bisphosphonates may be modified. Plasma clearance is rapid (half-life around 2 hours) because of the rapid uptake of 20-60% of the absorbed fraction into the skeleton. The remainder is excreted in the urine. The half-life in bone is very long, with release of bisphosphonates occurring only after resorption of bone into which the compounds have been taken up.
The strong affinity of bisphosphonates, and other hydroxyapatite-targeting moieties, for bone enables the polymers of the invention to be used as hydroxyapatite-targeting delivery systems for biologically active agents. Preferably, the isolatable, activated hydroxyapatite-targeting polymers can be efficiently attached to the biologically active agent in a single step.
As noted above, the hydroxyapatite-targeting polymeric structure of the invention has at least one terminus bonded to a protected or unprotected chemically reactive group. The chemically reactive group is preferably suitable for attachment to a finctional group on a biologically active agent. Examples of suitable chemically reactive groups include hydroxyl, protected hydroxyl, active ester, active carbonate, acetal, aldehyde, aldehyde hydrates, alkenyl, acrylate, methacrylate, acrylamide, active sulfone, amine, protected amine, hydrazide, protected hydrazide, thiol, protected thiol, carboxylic acid, protected carboxylic acid, isocyanate, isothiocyanate, maleimide, vinylsulfone, dithiopyridine, vinylpyridine, iodoacetamide, epoxide, glyoxals, diones, mesylates, tosylates, and tresylate.
As would be understood in the art, the term xe2x80x9cprotectedxe2x80x9d refers to the presence of a protecting group or moiety that prevents reaction of the chemically reactive group under certain reaction conditions. The protecting group will vary depending on the type of chemically reactive group being protected. For example, if the chemically reactive group is an amine or a hydrazide, the protecting group is preferably selected from the group of tert-butyloxycarbonyl (t-Boc) and 9-fluorenylmethoxycarbonyl (Fmoc). If the chemically reactive group is a thiol, the protecting group is preferably orthopyridyldisulfide. If the chemically reactive group is a carboxylic acid, such as butanoic or propionic acid, or a hydroxyl group, the protecting group is preferably benzyl. Other protecting groups known in the art may also be used in the invention.
In one embodiment, the chemically reactive groups are selected from the group consisting of hydroxyl, protected hydroxyl, amine, protected amine, carboxylic acid, protected carboxylic acid, maleimide, active carbonates, such as N-hydroxysuccinimidyl carbonates and 1-benzotriazolyl carbonates, and active esters, such as N-hydroxysuccinimidyl esters and 1-benzotriazolyl esters.
One useful hydroxyapatite-targeting polymer has the following general structure:
Qxe2x80x94POLYxe2x80x94Lxe2x80x94T
wherein POLY is a water-soluble and non-peptidic polymer, Q is a protected or unprotected chemically reactive group, L is a linker, and T is a hydroxyapatite-targeting moiety. Preferably, POLY is a poly(alkylene glycol), such as poly(ethylene glycol), having an average molecular weight from about 200 Da to about 100,000 Da, and T is a bisphosphonate. The linker L is the residue of the functional group used to attach the hydroxyapatite-targeting moiety to the polymer backbone. For example, the linker L may be a hydrolytically stable linkage selected from the group consisting of ether linkages, thio-ether linkages, amide linkages, amine linkages, urea linkages, or carbamate linkages. The linker L may also be a degradable linkage as described in more detail below.
In another embodiment, the hydroxyapatite-targeting polymer has the following general structure:
Qxe2x80x94PEGxe2x80x94Lxe2x80x94T
wherein PEG is a poly(ethylene glycol) having an average molecular weight from about 200 Da to about 100,000 Da, Q is a protected or unprotected chemically reactive group, L is a linker, and T is a bisphosphonate. Advantageously, PEG is xe2x80x94CH2CH2Oxe2x80x94(CH2CH2O)nxe2x80x94CH2CH2xe2x80x94, wherein n is about 3 to about 2000.
In a third embodiment, the hydroxyapatite-targeting polymer has the following general structure:
Txe2x80x94Lxe2x80x94POLYaxe2x80x94R(xe2x80x94POLYbxe2x80x94X)q
wherein POLYa and POLYb are water-soluble and non-peptidic polymer backbones that may be the same or different;
each X is independently selected from the group consisting of alkoxy, substituted alkoxy, aryloxy, substituted aryloxy, hydroxyl, protected hydroxyl, active ester, active carbonate, acetal, aldehyde, aldehyde hydrates, alkenyl, acrylate, methacrylate, acrylamide, active sulfone, amine, protected amine, hydrazide, protected hydrazide, thiol, protected thiol, carboxylic acid, protected carboxylic acid, isocyanate, isothiocyanate, maleimide, vinylsulfone, dithiopyridine, vinylpyridine, iodoacetamide, epoxide, glyoxals, diones, mesylates, tosylates, tresylate, and xe2x80x94Lxe2x80x94T, with the proviso that at least one X is not xe2x80x94Lxe2x80x94T;
R is a central core molecule, such as an amino acid or a polyol;
L is a linker;
T is a hydroxyapatite-targeting moiety; and
q is an integer from 2 to about 300.
Preferably, POLYa and POLYb are both poly(ethylene glycol) and R is selected from the group consisting of trimethylolpropane, di-rimethylolpropane, glycerol, pentaerythritol, sorbitol, lysine, and di-lysine.
Some examples of preferred embodiments of the hydroxyapatite-targeting polymers of the invention are provided as follows:
PEG(2,000)-xcex1-amine-xcfx89-AHPDP,
PEG(2,000)-xcex1-N-maleimido-xcfx89-AHPDP,
PEG(2,000)-xcex1-AHPDP-xcfx89-propionic acid,
PEG(2,000)-xcex1-AHPDP-xcfx89-propionic acid, N-hydroxysuccinimide ester,
PEG(2,000)-xcex1-AHPDP-xcfx89-protein,
PEG(2,000)-xcex1-L-tyrosine-xcfx89-AHPDP, and
PEG(10,000)-(xcex1-AHPDP)4 
where xe2x80x94AHPDP represents 3-amino-1-hydroxypropane-1,1-diphosphonic acid.
The invention also includes conjugates of the hydroxyapatite-targeting polymers of the invention and biologically active agents. For example, the invention provides hydroxyapatite-targeting, biologically active polymers of the following general structure:
Dxe2x80x94Lxe2x80x2xe2x80x94POLYxe2x80x94Lxe2x80x94T
wherein POLY is a water-soluble and non-peptidic polymer, D is a biologically active agent, L and Lxe2x80x2 are linkers which may be the same or different, and T is a hydroxyapatite-targeting moiety.
In another embodiment, the invention provides hydroxyapatite-targeting, biologically active polymers of the following general structure:
Dxe2x80x94Lxe2x80x2xe2x80x94PEGxe2x80x94Lxe2x80x94T
wherein PEG is a poly(ethylene glycol) having an average molecular weight from about 200 Da to about 100,000 Da, D is a biologically active agent, L and Lxe2x80x2 are linkers which may be the same or different, and T is a bisphosphonate.
Further, the invention includes hydroxyapatite-targeting, biologically active polymers of the following general structure:
Txe2x80x94Lxe2x80x94POLYaxe2x80x94R(xe2x80x94POLYbxe2x80x94Lxe2x80x2xe2x80x94A)q
wherein POLYa and POLYb are water-soluble and non-peptidic polymer backbones that may be the same or different;
each A is independently selected from the group consisting of hydroxyapatite-targeting moieties and biologically active agents, with the proviso that at least one A is a biologically active agent;
R is a central core molecule such as an amino acid or polyol;
L and Lxe2x80x2 are linkers which may be the same or different;
T is a hydroxyapatite-targeting moiety; and
q is an integer from 2 to about 300.
A method of utilizing a hydroxyapatite surface, such as bone, as a reservoir for a releasable biologically active agent is also provided by the invention. The method includes providing a hydroxyapatite-targeting, biologically active polymeric structure comprising a linear or branched water-soluble and non-peptidic polymer backbone having at least two termini, a first terminus being covalently bonded to a hydroxyapatite-targeting moiety and a second terminus covalently bonded to a biologically active agent through a linker, wherein at least one of the polymer backbone and the linker comprises a hydrolytically or enzymatically degradable linkage. For example, in any of the structures given above for the biologically active polymers, the L, Lxe2x80x2, POLY or PEG moieties may include a hydrolytically or enzymatically degradable linkage therein. For example, PEG can be prepared with ester linkages in the polymer backbone that are subject to hydrolysis. This hydrolysis results in cleavage of the polymer into fragments of lower molecular weight, as shown below.
xe2x80x94PEGxe2x80x94CO2xe2x80x94PEG+H2Oxe2x80x94PEGxe2x80x94CO2H+HOxe2x80x94PEGxe2x80x94
Preferably, the hydrolytically or enzymatically degradable linkage is selected from the group consisting of imine, carbonate, carboxylate ester, phosphoester, orthoester, acetal, carbamate linkages of the formula 
where Ar is an aryl group, disulfide, and peptide. However, other degradable linkages known in the art may be used.
A therapeutically effective amount of the polymeric structure is administered to, for example, a bone-containing mammal, such that at least a portion of the polymeric structure is bonded to a bone surface by the hydroxyapatite-targeting moiety. The hydroxyapatite-targeting polymeric structure, with the releasable biologically active agent attached thereto, will initially target bone surfaces within the organism, thereby using the bone surface as a reservoir or depot. Due to the presence of the degradable linkage in either the polymer backbone or the linker, the biologically active agent will be released into the organism over time as the linkage degrades. In this manner, the biologically active agent is delivered to other parts of the organism for treatment of disease or other medical conditions. The released biologically active agent may be in native form, or attached to a linker or the linker and a fragment of the polymer backbone, depending on the placement of the degradable linkage. By tethering or anchoring the biologically active agent to bone, the residence time of the biologically active agent can be increased, which can lead to increased treatment efficacy.
The xe2x80x9ctherapeutically effective amountxe2x80x9d will depend upon a number of factors, including the nature and severity of the condition or disease being treated, the type of biologically active agent being used, the size, age and general health of the subject, and other factors.
The polymeric structure may be administered by various routes, including oral, pulmonary, intravenous, subcutaneous, intramuscular, buccal, nasal, ocular, and rectal. The polymeric structure may also be administered with one or more pharmaceutically acceptable carriers, excipients or diluents.
The hydroxyapatite-targeting polymers of the invention can also be used to bind to other calcium phosphate surfaces or coatings, including hydroxyapatite-coated prosthetic devices and calcium phosphate particles. For example, the hydroxyapatite-targeting polymers of the invention can be used to prevent protein and cell adsorption into prosthetic devices or as part of a synthetically produced delivery device for biologically active agents.
The invention also includes methods of preparing the hydroxyapatite-targeting polymers, and biologically active conjugates thereof, described above. In a preferred embodiment, the activated polymers are prepared by providing a polymeric structure comprising a linear or branched water-soluble and non-peptidic polymer backbone having at least two termini, a first terminus covalently bonded to a first protected chemically reactive group and a second terminus covalently bonded to a second chemically reactive group selected from the group consisting of active carbonates and active esters. Examples of suitable active carbonates and active esters include N-hydroxysuccinimidyl esters, 1-benzotriazolyl esters, N-hydroxysuccinimidyl carbonates and 1-benzotriazolyl carbonates. The second chemically reactive group is reacted with a hydroxyapatite-targeting moiety to form a hydroxyapatite-targeting polymeric structure. The first protected chemically reactive group is preferably selected from the group consisting of protected hydroxyl, protected amine, protected carboxylic acid, protected hydrazide, and protected thiol. In this manner, at least one terminus of the polymer is protected such that the hydroxyapatite-targeting moiety does not attach thereto. This ensures that at least one terminus will be available for subsequent reaction with a biologically active agent. In one embodiment, the following polymer structure is used to react with the hydroxyapatite-targeting moiety:
Xxe2x80x94Oxe2x80x94CH2CH2Oxe2x80x94(CH2CH2O)nxe2x80x94CH2CH2xe2x80x94CO2Yxe2x80x2
wherein X is an active carbonate or an active ester, Yxe2x80x2 is benzyl, and n is about 3 to about 2000. The method may further include deprotecting the first protected chemically reactive group so that the chemically reactive group is available for reaction, for example with a biologically active agent.
The method of producing a hydroxyapatite-targeting, biologically active polymer preferably includes providing a polymeric structure comprising a linear or branched water-soluble and non-peptidic polymer backbone having at least two termini, a first terminus being covalently bonded to a hydroxyapatite-targeting moiety and a second terminus covalently bonded to a chemically reactive group. The chemically reactive group is reacted with a biologically active agent such that the biologically active agent is attached to the second terminus of the polymeric structure to form a hydroxyapatite-targeting, biologically active polymeric structure. As would be understood by one of ordinary skill, the choice of chemically reactive group will depend on the available functional groups on the biologically active agent. For example, if the biologically active agent contains amine, hydroxyl, or thiol functional groups, an active carbonate or active ester may be used as the chemically reactive group. Particularly preferred chemically reactive groups useful for reacting with biologically active moieties include hydroxyl, amine, carboxylic acid, maleimide, active carbonates and active esters. The biologically active agent preferably comprises peptides, proteins, enzymes, small molecule drugs, dyes, nucleosides, oligonucleotides, lipids, phospholipids, cells, viruses, liposomes, microparticles or micelles. In one embodiment, the biologically active agent has at least one hydroxy group, such as quinidine, camptothecan, and paclitaxel.